1. Field of the Invention
The invention relates to a class of peptides which disrupts mitotic function in cells.
2. Background of the Invention
Early seed development in angiosperms is characterized by rapid cell division and differentiation followed by cessation of cell division and the start of cell expansion during which DNA endoreduplication and massive synthesis of storage proteins, carbohydrates and lipids occur in the endosperm of cereals and in the cotyledons of legumes (1). The molecular mechanisms underlying these early events are poorly understood. The temporal expression of 2S albumins, a diverse group of water soluble proteins found in developing seeds, has been shown to coincide with the initiation of cell expansion after the cells stopped dividing in developing pea embryos (2). In situ hybridization work on Arabidopsis involving actively dividing and nondividing parynchema cells also suggests the possible existence of a regulatory mechanism that is common to 2S albumin gene expression and mitotic activity (3). However, the functional role of 2S albumins in regulation of mitosis during seed development remains to be established.
The invention provides methods and compositions of selectively modulating mitotic function in a target cell demonstrating undesirable mitotic function. Suitable target cells include mammalian, plant and bacterial cells, which cells may be in vitro or in situ. The general methods involve introducing into the target cell an effective amount of a modulator of mitotic function comprising a Gm2S-1 peptide, and/or contiguous acidic amino acids, such as Asp or Glu, whereby the undesirable mitotic function of the cell is selectively modulated. In particular embodiments, the Gm2S-1 peptide comprises the contiguous acidic amino acids and/or comprises residues 33-43, residues 85-91, residues 123-127, residues 85-127, at least 12 contiguous residues of SEQ ID NO:2, residues 1-35 and/or SEQ ID NO:2. The peptide may be introduced by transfecting the cell with a nucleic acid which encodes the peptide or comprises SEQ ID NO:1 or a fragment thereof which modulates the expression of a resident Gm2s-1 peptide-encoding gene or transcript.
The invention encompasses a variety of compositions which may be used in the subject methods including modulators of mitotic function, Gm2S-1 peptides, Gm2S-1 peptide-encoding nucleic acids, Gm2S-1 peptide-specific binding agents such as antibodies, and nucleic acid hybridization probes and primers comprising a strand of SEQ ID NO:1 or a fragment thereof sufficient to specifically hybridize with and thereby facilitate identifying, cloning, amplifying and/or modulating the expression of a Gm2S-1 peptide-encoding gene.
The invention provides methods and compositions of selectively disrupting mitotic function in a target cell demonstrating undesirable mitotic function. Hence, the methods may be used to interfere with (e.g. promote, prevent or delay) targeted cell division. The invention is applicable to a wide variety of indications where mitotic function is undesirable quantitatively, qualitatively, spacially, temporally, etc. For example, in one particular embodiment, the methods and compositions are used to control undesirable growth of cells in human neoplasia, such as cancer, restinosis, etc. In another embodiment, the invention is used to prevent the normal division of harmful micororganisms, e.g. pathogenic bacteria such as described in Medical Microbiology 4th Ed. (S. Baron, Ed., 1996, UT Med Branch at Galveston). In yet another embodiment, the invention is used to regulate plant seed development by controling the timing of the termination of cell division that allows DNA endoreduplication to occur.
The general methods involve introducing into the target cell an effective amount of a peptide modulator, preferably a disruptor, of mitotic function comprising contiguous acidic amino acids, preferably at least 6, more preferably at least 7, more preferably at least 8 such as Asp or Glu, sufficient to selectively modulate mitotic function of a cell. As demonstrated herein, the invention encompasses a wide variety of suitable methods, amounts, and peptide lengths and compositions, which are readily optimized empirically.
In particular embodiments, the modulator peptide comprises a Gm2S-1 peptide which comprises at least 5, preferably at least 6, more preferably at least 7 of the contiguous acidic amino acids. In other preferred embodiments, the peptide comprise a Gm2S-1 peptide comprising at least an 8, preferably at least a 10, more preferably at least a 20 residue domain of SEQ ID NO:2 sufficient to selectively modulate mitotic function of the cell. Such peptides may derive from the Gm2s-1 signal peptide (residues 01-21 of SEQ ID NO:2), lunasin (residues 22-64 of SEQ ID NO:2), the Gm2S-1 linker peptide (residues 65-81 of SEQ ID NO:2) or alisin (residues SEQ ID NO:2, residues 82-158). In other preferred embodiments, the peptide comprises residues 33-43 of SEQ ID NO:2, residues 85-91 of SEQ ID NO:2, residues 123-127 of SEQ ID NO:2, residues 85-127 of SEQ ID NO:2 and/or at least 12 contiguous residues of SEQ ID NO:2, residues 1-35. The contiguous acidic amino acids are preferably proximate to (i.e. within 12 residues, preferably within 6 residues, more preferably within 3 residues) or at the C-terminus of the modulator. The modulator may comprise a wide variety of additional moieties, especially moities positioned N-terminally relative to the acidic amino acids, including moieties which provide for detection, targeting, stability, proteolytic resistance, etc.
The subject modulators comprising Gm2S-1 peptides provide Gm2S-1 peptide specific activity or function, such as Gm2S-1-specific disruption of mitotic function, ligand/antibody binding or binding inhibitory, immunogenicity, etc. Gm2S-1 peptide-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in,animals (e.g. gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the molecular interaction of a Gm2S-1 peptide with a binding target is evaluated. The binding target may be a natural intracellular binding target such as a Gm2S-1 peptide regulating protein (e.g. mapmodulin, Ulitzur et al., 1997, PNAS 94, 5084-5089) or other regulator that directly modulates a Gm2S-1 peptide activity or its localization; or non-natural binding target such a specific immune protein such as an antibody, or an Gm2S-1 peptide specific agent such as those identified in bio/chemical screening assays. Gm2S-1 peptide-binding specificity may assayed by mitotic disruption assays described below, binding equilibrium constants (usually at least about 107 Mxe2x88x921, preferably at least about 108 Mxe2x88x921, more preferably at least about 109 Mxe2x88x921), by the ability of the subject peptides to function as negative mutants in a Gm2S-1 peptide-expressing cells, to elicit a Gm2S-1 peptide specific antibody in a heterologous host (e.g. a rodent or rabbit), etc. The Gm2S-1 peptide binding specificity of preferred Gm2S-1 peptides necessarily distinguishes that of the tubulin, MAPs and Mapmodulin.
In particular embodiments, modulators comprising Gm2S-1 peptides are isolated or pure: an xe2x80x9cisolatedxe2x80x9d peptide is unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, and more preferably at least about 5% by weight of the total peptide in a given sample and a pure peptide constitutes at least about 90%, and preferably at least about 99% by weight of the total peptide in a given sample. The peptides may be synthesized, produced by recombinant technology, or purified from cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) or that are otherwise known in the art. Material and methods for the expression of heterologous recombinant peptides in bacterial cells (e.g. E. coli), yeast (e.g. S. Cerevisiae), animal cells (e.g. CHO, 3T3, BHK, baculovirus-compatible insect cells, etc.). The peptides may be provided uncomplexed with other peptide, complexed in a wide variety of non-covalent associations and binding complexes, complexed covalently with other Gm2S-1 or non-Gm2S-1 peptide sequences (homo or hetero-chimeric peptides), etc.
The invention provides binding agents specific to the claimed modulators, including substrates, agonists, antagonists, natural intracellular binding targets, etc., methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. For example, novel peptide-specific binding agents include Gm2S-1 peptide -specific receptors, such as somatically recombined polypeptide receptors like specific antibodies or T-cell antigen receptors (see, e.g Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory) and other natural intracellular binding agents identified with assays such as one-, two- and three-hybrid screens, non-natural intracellular binding agents identified in screens of chemical libraries, etc. Agents of particular interest modulate Gm2S-1 peptide function, e.g. Gm2S-1 peptide-dependent mitotic disruption.
The invention also provides efficient methods of identifying agents active at the level of a Gm2S-1 modulatable cellular function. Generally, these screening methods involve assaying for compounds which modulate a Gm2S-1 peptide interaction with a natural Gm2S-1 peptide binding target, etc. A wide variety of assays for binding agents are provided including labeled in vitro protein-protein binding assays, immunoassays, cell based assays, etc. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. Agents that modulate the interactions of a Gm2S-1 peptide with its ligands/natural binding targets can be used to modulate biological processes associated a Gm2S-1 peptide function, e.g. by contacting a cell comprising a Gm2S-1 peptide (e.g. administering to a subject comprising such a cell) with such an agent. Biological processes mediated by Gm2S-1 peptides include a wide variety of cellular events which are mediated when a Gm2S-1 peptide binds a ligand e.g. plant seed growth regulation.
The amino acid sequences of the subject peptides are used to back-translate peptide-encoding nucleic acids optimized for selected expression systems (Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural Gm2S-1 peptide-encoding nucleic acid sequences (xe2x80x9cGCGxe2x80x9d software, Genetics Computer Group, Inc, Madison Wis.). Modulator peptide-encoding nucleic acids are used in peptide-expression vectors and incorporated into recombinant host cells, e.g. for expression and screening, e.g. for functional studies such as the efficacy of candidate agents to manipulate modulator peptide-modulated cell function, etc.
The invention also provides Gm2S-1 nucleic acids including hybridization probes and replication/amplification primers having a Gm2S-1 cDNA specific sequence comprising a fragment of a strand of SEQ ID NO:1 sufficient to effect specific hybridization to the complementary strand of SEQ ID NO:1 (i.e. specifically hybridize with a nucleic acid comprising the corresponding opposite strand of SEQ ID NO:1, in the presence of a soybean seed midmaturation library cDNA library). Such primers or probes are at least 12, preferably at least 24, more preferably at least 36 and most preferably at least 96 bases in length. Demonstrating specific hybridization generally requires stringent conditions, i.e. those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium titrate/0.1% SDS at 50xc2x0 C., or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42xc2x0 C. Another example is use of 50% formamide, 5xc3x97SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5xc3x97Denhardt""s solution, sonicated salmon sperm DNA (50 (g/ml), 0.1% SDS, and 10% dextran sulfate at 42xc2x0 C., with washes at 42xc2x0 C. in 0.2xc3x97SSC and 0.1% SDS. Gm2S-1 nucleic acids can also be distinguished using alignment algorithms, such as BLASTX (Altschul et al. (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410). In one embodiment, the subject Gm2S-1 nucleic acids comprise Gm2S-1 peptide encoding regions of SEQ ID NO:1; e.g. a Gm2S-1 signal peptide is encoded by bases 17-79, a lunasin peptide by bases 80-208, a lunasin-alisin linker peptide by bases 209-259, an alisin peptide by bases 260-490.
The subject nucleic acids are of synthetic/non-natural sequences and/or are isolated, i.e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. Recombinant nucleic acids comprising the nucleotide sequence of SEQ ID NO:1, or the subject fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by (i.e. contiguous with) a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, which is at a terminus or is immediately flanked by a sequence other than that which it is joined to on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc.
The subject nucleic acids find a wide variety of applications including use as translatable transcripts, knock-in/out vectors, hybridization probes, PCR primers, diagnostic nucleic acids, etc.; use in detecting the presence of Gm2S-1 genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional Gm2S-1 homologs and structural analogs. In diagnosis, Gm2S-1 hybridization probes find use in identifying wild-type and mutant Gm2S-1 alleles. Gm2S-1 nucleic acids are used to effect and/or modulate cellular expression or intracellular concentration or availability of active Gm2S-1. Methods for effecting the targeted expression of genes encoding the subject modulators are known in the art; see, e.g. Altenschmidt et al., 1997, J Mol Med 75:259-266; Perales et al. 1997, Proc Natl Acad Sci USA 94:6450-6455; Schmidt et al., 1997, Gene 190:211-216; Oldfield et al., 1993, Human Gene Therapy 4: 39-46; Asgari et al., 1997, Int J. Cancer 71:377-382; He D; et al. 1997, Cancer Res 57:1868-1872. In a particular embodiment, the subject Gm2S-1 peptide is introduced by transfecting the cell with a nucleic acid encoding the peptide particularly, wherein the nucleic acid comprises. SEQ ID NO:1 or a fragment thereof. Therapeutic nucleic acid compositions may be advantageously combined and/or used in combination with other therapeutic or prophylactic agents, different from the subject compounds. In many instances, administration in conjunction with the subject compositions enhances the efficacy of such agents, see e.g. Goodman and Gilman""s The Pharmacological Basis of Therapeutics, 9th Ed., 1996, McGraw-Hill.